1. Field of the Invention
The present invention relates, in general, to a method of producing L-amino acids and to a gene encoding phosphoglucoisomerase.
2. Background Information
Bacterial cells are used industrially to produce amino acids by fermentation processes (Ishino, S. et al., J. Gen. Appl. Microbiol. 37:157-165 (1991), Kinoshita, S., Nakayama, K. and Nagasaki, S., J. Gen. Appl. Microbiol. 4:128-129 (1958)). Although numerous research reports and reviews have appeared concerning fermentation processes and the mechanisms of accumulation of amino acids, more progress needs to be made to increase the yields of amino acids from microorganisms (Ishino, S. et al., J. Gen. Appl. Microbiol. 37:157-165 (1991), Aida, K. et al., eds., xe2x80x9cBiotechnology of Amino Acid Production,xe2x80x9d Kodansha (Tokyo)/Elsevier (New York) (1986) and Marx, A. et al., Metabolic Engineering 1:35-48 (1999)).
There has been some success in using metabolic engineering to direct the flux of glucose derived carbons toward aromatic amino acid formation (Flores, N. et al., Nature Biotechnol. 14:620-623 (1996)). However, the successful application in producer strains has not yet been documented (Berry, A., TIBTECH 14:250-256 (1996)).
Metabolic engineering relates to manipulation of the flow of carbons of starting materials, such as carbohydrates and organic acids, through the variety of metabolic pathways during fermentation. Studies have been done, for example, on the central metabolism of Corynebacterium glutamicum using 13C NMR studies (Ishino, S. et al., J. Gen. Appl. Microbiol. 37:157-165 (1991), Marx, A. et al., Biotechnology and Bioengineering 49:111-129 (1996)). Additionally, also using 13C NMR, Walker et al. (Walker, T. et al, J. Biol. Chem. 257:1189-1195 (1982)) analyzed glutamic acid fermentation by Microbacterium ammoniaphilum, and Inbar et al (Inbar, L. et al., Eur. J. Biochem. 149:601-607 (1985)) studied lysine fermentation by Brevibacterium flavum. 
The present invention solves a problem of improving yields of amino acids during fermentation using metabolic engineering.
The present invention provides a method of producing L-amino acids by culturing altered bacterial cells having increased amounts of NADPH as compared to unaltered bacterial cells, whereby L-amino acid yields from said altered bacterial cells are greater than yields from unaltered bacterial cells.
The present invention also provides a method of producing a bacterial cell with a mutated phosphoglucose isomerase (pgi) gene comprising (a) subcloning an internal region of the pgi gene into a suicide vector; and (b) inserting said suicide vector into a bacterial genome, via homologous recombination, whereby a bacterial cell with an altered pgi gene is produced. The invention further provides an altered bacterial cell produced according to this method.
The invention also provides a vector useful according to this method.
The present invention further provides isolated nucleic acid molecules comprising a polynucleotide encoding the Corynebacterium glutamicum phosphoglucose isomerase polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or one of the amino acid sequence encoded by the DNA clone deposited in a bacterial host as NRRL Deposit Number B-30174 on Aug. 17, 1999.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of Pgi polypeptides or peptides by recombinant techniques.
The invention further provides an isolated Pgi peptide having an amino acid sequence encoded by a polynucleotide described herein.
Further advantages of the present invention will be clear from the description that follows.